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1.
Med J Malaysia ; 74(4): 347-348, 2019 08.
Article in English | MEDLINE | ID: mdl-31424049

ABSTRACT

Varicella zoster infection is one of the self-limiting viral infections during childhood and dengue fever is an endemic infection in Malaysia, which commonly occurs in the form of nonspecific febrile illness at the initial stage. It is rare for the two viral infections to occur simultaneously. A case of dengue fever without warning sign in a five-year old girl was reported, with early symptoms of fever and vesicular rashes. She was clinically diagnosed with varicella zoster infection during the first visit. Surprisingly, she remained febrile even on day six of illness despite no new vesicular lesions on her skin. Due to suspicion of another infection, follow-up investigation was done and revealed isolated thrombocytopenia. This finding was confirmed with positive NS1Ag. A case of rare dengue fever concomitant with varicella zoster infection was reported.


Subject(s)
Chickenpox/diagnosis , Coinfection/diagnosis , Dengue/diagnosis , Chickenpox/complications , Child, Preschool , Dengue/complications , Female , Humans
2.
Biochem J ; 458(3): 575-83, 2014 Mar 15.
Article in English | MEDLINE | ID: mdl-24341532

ABSTRACT

The proposed Ca2+-activated Cl- channel protein Best1 (bestrophin 1) is expressed and functionally important in the retina and in the brain. Human BEST1 has two known splice variants, Best1V1 and Best1V2, which arise from alternative splicing of two exons: exon 2 splicing results in a unique N-terminal domain, whereas alternative splicing of exon 11 produces two mutually exclusive C-termini. Prior studies were limited to Best1V1 and its clinically relevant mutations. In the present work, we cloned a novel splice variant of Best1V1 missing exon 2 (Best1V1Δex2) and differing from each of the two previously identified isoforms by one alternatively spliced domain. This finding allowed us to determine the role for alternative splicing of the Best1 N- and C-termini. We heteroexpressed Best1V1Δex2 in HEK (human embryonic kidney)-293 cells, and compared its properties with Best1V1 and Best1V2. Western blot analysis confirmed protein expression from all three splice variants. Both Best1V1 and Best1V1Δex2 successfully formed Ca2+-activated Cl- channels, demonstrating that the N-terminus encoded by exon 2 is not essential for channel function. In contrast, Best1V2-expressing cells had no detectable Ca2+-activated Cl- currents, pointing to a critical role for splicing of the C-terminus. Surface protein biotinylation demonstrated that Best1V1 and Best1V1Δex2 are trafficked to the plasma membrane, whereas Best1V2 is not. These results define the impact of alternative splicing on Best1 function, and should be taken into consideration in future modelling of the Best1 protein structure.


Subject(s)
Alternative Splicing , Chloride Channels/genetics , Eye Proteins/genetics , Astrocytes/metabolism , Bestrophins , Cell Line, Tumor , Chloride Channels/metabolism , Cloning, Molecular , Exons , Eye Proteins/metabolism , Glioma/metabolism , HEK293 Cells , Humans , Neuroglia/metabolism , Protein Isoforms/metabolism
3.
Pflugers Arch ; 465(9): 1249-60, 2013 Sep.
Article in English | MEDLINE | ID: mdl-23515871

ABSTRACT

The Ca(2+) sensor stromal interacting molecule 1 (STIM1) and the Ca(2+) channel Orai1 mediate the ubiquitous store-operated Ca(2+) entry (SOCE) pathway activated by depletion of internal Ca(2+) stores and mediated through the highly Ca(2+)-selective, Ca(2+) release-activated Ca(2+) (CRAC) current. Furthermore, STIM1 and Orai1, along with Orai3, encode store-independent Ca(2+) currents regulated by either arachidonate or its metabolite, leukotriene C4. Orai channels are emerging as important contributors to numerous cell functions, including proliferation, migration, differentiation, and apoptosis. Recent studies suggest critical involvement of STIM/Orai proteins in controlling the development of several cancers, including malignancies of the breast, prostate, and cervix. Here, we quantitatively compared the magnitude of SOCE and the expression levels of STIM1 and Orai1 in non-malignant human primary astrocytes (HPA) and in primary human cell lines established from surgical samples of the brain tumor glioblastoma multiforme (GBM). Using Ca(2+) imaging, patch-clamp electrophysiology, pharmacological reagents, and gene silencing, we established that in GBM cells, SOCE and CRAC are mediated by STIM1 and Orai1. We further found that GBM cells show upregulation of SOCE and increased Orai1 levels compared to HPA. The functional significance of SOCE was evaluated by studying the effects of STIM1 and Orai1 knockdown on cell proliferation and invasion. Utilizing Matrigel assays, we demonstrated that in GBM, but not in HPA, downregulation of STIM1 and Orai1 caused a dramatic decrease in cell invasion. In contrast, the effects of STIM1 and Orai1 knockdown on GBM cell proliferation were marginal. Overall, these results demonstrate that STIM1 and Orai1 encode SOCE and CRAC currents and control invasion of GBM cells. Our work further supports the potential use of channels contributed by Orai isoforms as therapeutic targets in cancer.


Subject(s)
Brain Neoplasms/metabolism , Calcium Channels/metabolism , Calcium Signaling , Glioblastoma/metabolism , Membrane Proteins/metabolism , Neoplasm Proteins/metabolism , Action Potentials , Astrocytes/metabolism , Brain Neoplasms/pathology , Calcium/metabolism , Calcium Channels/genetics , Cell Line, Tumor , Cell Proliferation , Gene Expression Regulation, Neoplastic , Glioblastoma/pathology , Humans , Membrane Proteins/genetics , Neoplasm Invasiveness , Neoplasm Proteins/genetics , ORAI1 Protein , Stromal Interaction Molecule 1 , Transcription, Genetic , Up-Regulation
4.
Sci Signal ; 6(267): ra18, 2013 Mar 19.
Article in English | MEDLINE | ID: mdl-23512989

ABSTRACT

Endothelial barrier function is critical for tissue fluid homeostasis, and its disruption contributes to various pathologies, including inflammation and sepsis. Thrombin is an endogenous agonist that impairs endothelial barrier function. We showed that the thrombin-induced decrease in transendothelial electric resistance of cultured human endothelial cells required the endoplasmic reticulum-localized, calcium-sensing protein stromal interacting molecule 1 (STIM1), but was independent of Ca2+ entry across the plasma membrane and the Ca2+ release-activated Ca2+ channel protein Orai1, which is the target of STIM1 in the store-operated calcium entry pathway. We found that STIM1 coupled the thrombin receptor to activation of the guanosine triphosphatase RhoA, stimulation of myosin light chain phosphorylation, formation of actin stress fibers, and loss of cell-cell adhesion. Thus, STIM1 functions in pathways that are dependent on and independent of Ca2+ entry.


Subject(s)
Calcium Channels/metabolism , Calcium/metabolism , Endothelial Cells/metabolism , Membrane Proteins/metabolism , Neoplasm Proteins/metabolism , Blotting, Western , Cells, Cultured , Electric Impedance , Endoplasmic Reticulum/metabolism , Endothelial Cells/drug effects , Endothelial Cells/physiology , Fluorescent Antibody Technique , HEK293 Cells , Human Umbilical Vein Endothelial Cells/drug effects , Human Umbilical Vein Endothelial Cells/metabolism , Human Umbilical Vein Endothelial Cells/physiology , Humans , Membrane Proteins/genetics , Myosin Light Chains/metabolism , Myosin-Light-Chain Kinase/genetics , Myosin-Light-Chain Kinase/metabolism , Neoplasm Proteins/genetics , ORAI1 Protein , Phosphorylation , RNA Interference , Signal Transduction/genetics , Signal Transduction/physiology , Stromal Interaction Molecule 1 , TRPC Cation Channels/genetics , TRPC Cation Channels/metabolism , TRPC6 Cation Channel , Thrombin/pharmacology , rhoA GTP-Binding Protein/genetics , rhoA GTP-Binding Protein/metabolism
5.
PLoS One ; 5(8): e12304, 2010 Aug 20.
Article in English | MEDLINE | ID: mdl-20808839

ABSTRACT

Gliomas are morbid brain tumors that are extremely resistant to available chemotherapy and radiology treatments. Some studies have suggested that calcium-activated potassium channels contribute to the high proliferative potential of tumor cells, including gliomas. However, other publications demonstrated no role for these channels or even assigned them antitumorogenic properties. In this work we characterized the expression and functional contribution to proliferation of Ca(2+)-activated K(+) channels in human glioblastoma cells. Quantitative RT-PCR detected transcripts for the big conductance (BK), intermediate conductance (IK1), and small conductance (SK2) K(+) channels in two glioblastoma-derived cell lines and a surgical sample of glioblastoma multiforme. Functional expression of BK and IK1 in U251 and U87 glioma cell lines and primary glioma cultures was verified using whole-cell electrophysiological recordings. Inhibitors of BK (paxilline and penitrem A) and IK1 channels (clotrimazole and TRAM-34) reduced U251 and U87 proliferation in an additive fashion, while the selective blocker of SK channels UCL1848 had no effect. However, the antiproliferative properties of BK and IK1 inhibitors were seen at concentrations that were higher than those necessary to inhibit channel activity. To verify specificity of pharmacological agents, we downregulated BK and IK1 channels in U251 cells using gene-specific siRNAs. Although siRNA knockdowns caused strong reductions in the BK and IK1 current densities, neither single nor double gene silencing significantly affected rates of proliferation. Taken together, these results suggest that Ca(2+)-activated K(+) channels do not play a critical role in proliferation of glioma cells and that the effects of pharmacological inhibitors occur through their off-target actions.


Subject(s)
Gene Expression Regulation, Neoplastic , Glioblastoma/genetics , Glioblastoma/pathology , Intermediate-Conductance Calcium-Activated Potassium Channels/genetics , Intermediate-Conductance Calcium-Activated Potassium Channels/metabolism , Large-Conductance Calcium-Activated Potassium Channels/genetics , Large-Conductance Calcium-Activated Potassium Channels/metabolism , Amino Acid Sequence , Cell Line, Tumor , Cell Proliferation/drug effects , Down-Regulation/genetics , Electric Conductivity , Gene Expression Regulation, Neoplastic/drug effects , Gene Knockdown Techniques , Glioblastoma/surgery , Humans , Intermediate-Conductance Calcium-Activated Potassium Channels/antagonists & inhibitors , Intermediate-Conductance Calcium-Activated Potassium Channels/deficiency , Large-Conductance Calcium-Activated Potassium Channels/antagonists & inhibitors , Large-Conductance Calcium-Activated Potassium Channels/deficiency , Molecular Sequence Data , Potassium/metabolism , Potassium Channel Blockers/pharmacology , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Small Interfering/genetics , Small-Conductance Calcium-Activated Potassium Channels/genetics
6.
J Biol Chem ; 285(28): 21303-12, 2010 Jul 09.
Article in English | MEDLINE | ID: mdl-20442409

ABSTRACT

Multiple Ca(2+) release and entry mechanisms and potential cytoskeletal targets have been implicated in vascular endothelial barrier dysfunction; however, the immediate downstream effectors of Ca(2+) signals in the regulation of endothelial permeability still remain unclear. In the present study, we evaluated the contribution of multifunctional Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) as a mediator of thrombin-stimulated increases in human umbilical vein endothelial cell (HUVEC) monolayer permeability. For the first time, we identified the CaMKIIdelta(6) isoform as the predominant CaMKII isoform expressed in endothelium. As little as 2.5 nM thrombin maximally increased CaMKIIdelta(6) activation assessed by Thr(287) autophosphorylation. Electroporation of siRNA targeting endogenous CaMKIIdelta (siCaMKIIdelta) suppressed expression of the kinase by >80% and significantly inhibited 2.5 nM thrombin-induced increases in monolayer permeability assessed by electrical cell-substrate impedance sensing (ECIS). siCaMKIIdelta inhibited 2.5 nM thrombin-induced activation of RhoA, but had no effect on thrombin-induced ERK1/2 activation. Although Rho kinase inhibition strongly suppressed thrombin-induced HUVEC hyperpermeability, inhibiting ERK1/2 activation had no effect. In contrast to previous reports, these results indicate that thrombin-induced ERK1/2 activation in endothelial cells is not mediated by CaMKII and is not involved in endothelial barrier hyperpermeability. Instead, CaMKIIdelta(6) mediates thrombin-induced HUVEC barrier dysfunction through RhoA/Rho kinase as downstream intermediates. Moreover, the relative contribution of the CaMKIIdelta(6)/RhoA pathway(s) diminished with increasing thrombin stimulation, indicating recruitment of alternative signaling pathways mediating endothelial barrier dysfunction, dependent upon thrombin concentration.


Subject(s)
Calcium-Calmodulin-Dependent Protein Kinase Type 2/metabolism , Endothelium/pathology , Gene Expression Regulation, Enzymologic , Thrombin/metabolism , rhoA GTP-Binding Protein/metabolism , Animals , Calcium/metabolism , Endothelial Cells/cytology , Endothelium/metabolism , Endothelium, Vascular/cytology , Extracellular Signal-Regulated MAP Kinases/metabolism , Humans , Male , Models, Biological , Protein Isoforms , Rats , Rats, Sprague-Dawley , Thrombin/chemistry
7.
J Biol Chem ; 285(25): 19173-83, 2010 Jun 18.
Article in English | MEDLINE | ID: mdl-20395295

ABSTRACT

Store-operated calcium (Ca(2+)) entry (SOCE) mediated by STIM/Orai proteins is a ubiquitous pathway that controls many important cell functions including proliferation and migration. STIM proteins are Ca(2+) sensors in the endoplasmic reticulum and Orai proteins are channels expressed at the plasma membrane. The fall in endoplasmic reticulum Ca(2+) causes translocation of STIM1 to subplasmalemmal puncta where they activate Orai1 channels that mediate the highly Ca(2+)-selective Ca(2+) release-activated Ca(2+) current (I(CRAC)). Whereas Orai1 has been clearly shown to encode SOCE channels in many cell types, the role of Orai2 and Orai3 in native SOCE pathways remains elusive. Here we analyzed SOCE in ten breast cell lines picked in an unbiased way. We used a combination of Ca(2+) imaging, pharmacology, patch clamp electrophysiology, and molecular knockdown to show that native SOCE and I(CRAC) in estrogen receptor-positive (ER(+)) breast cancer cell lines are mediated by STIM1/2 and Orai3 while estrogen receptor-negative (ER(-)) breast cancer cells use the canonical STIM1/Orai1 pathway. The ER(+) breast cancer cells represent the first example where the native SOCE pathway and I(CRAC) are mediated by Orai3. Future studies implicating Orai3 in ER(+) breast cancer progression might establish Orai3 as a selective target in therapy of ER(+) breast tumors.


Subject(s)
Breast Neoplasms/metabolism , Calcium Channels/metabolism , Gene Expression Regulation, Neoplastic , Receptors, Estrogen/metabolism , Calcium/metabolism , Cell Adhesion Molecules/metabolism , Cell Line, Tumor , Disease Progression , Endoplasmic Reticulum/metabolism , Humans , Membrane Proteins/metabolism , Neoplasm Proteins/metabolism , ORAI1 Protein , Patch-Clamp Techniques , Protein Transport , Stromal Interaction Molecule 1 , Stromal Interaction Molecule 2
8.
Transplant Proc ; 41(10): 4047-51, 2009 Dec.
Article in English | MEDLINE | ID: mdl-20005339

ABSTRACT

INTRODUCTION: The acceptance of a living donor kidney bearing cysts might implicate complications after the transplantation due to the natural history of renal cysts. We have presented our experience with transplantation of living donor kidneys containing cysts but not polycystic disease. PATIENTS AND METHODS: We retrospectively reviewed donor and recipient records of all living kidney transplants performed between January 1997 and April 2008. We analyzed serum creatinine and urea levels, as well as ultrasound scans concerning cyst size and morphology at hospital discharge as well as at 12 and 24 months after transplantation. RESULTS: Among 268 living kidney transplantations, we noted 25 donors with renal cysts. In the computed tomography scan reports, 19 kidneys were described to show a single and six, multiple cysts. The size of 10 single cysts was <5 mm; the other nine were a mean of 17.33 mm. Two of the multiple cyst kidneys had lesions <5 mm; in four kidneys, the mean cyst size was 27.25 mm. The renal function of the recipients was normal or almost normal at discharge with a tendency to lower levels at 12 and 24 months after transplantation. Ultrasound revealed changes in cyst diameter among 6/23 kidneys; the mean diameter increased after 12 months, namely, 8.25 mm to 11.5 mm after 24 months. The subgroup of patients with enlarged cysts showed creatinine and urea levels slightly higher than in the entire group. No aspects of malignancy were found, according to the Bosniak and Israel classification system. One suspicious cyst was tomographically confirmed to be hemorrhagic without any need for treatment. None of the living donors had any problems related to the donor nephrectomy or a need for dialysis due to renal insufficiency in the long term. In addition, the living donors who had even beforehand cystic lesions in their contralateral nonremoved kidney at the time of transplantation did not show complications upon follow-up. CONCLUSIONS: In our study, 25 living donor kidneys carried cysts. Neither cyst-related complications nor dysfunction of the transplanted organs occurred. An unroofing or excision of the cyst was generally not necessary. Regular ultrasound scans and optional computed tomography scans are recommended for follow-up. Based on this experience, we concluded that kidneys presenting cystic diseases should be considered to be suitable for transplantation without a hazard to the recipients, thus extending the pool of organs.


Subject(s)
Kidney Transplantation/methods , Living Donors , Nephrectomy/methods , Patient Selection , Polycystic Kidney Diseases/pathology , Adult , Aged , Creatinine/blood , Female , Follow-Up Studies , Functional Laterality , Humans , Male , Middle Aged , Polycystic Kidney Diseases/diagnostic imaging , Postoperative Complications/epidemiology , Radiography , Retrospective Studies , Time Factors , Tomography, Emission-Computed , Urea/blood
9.
Chirurg ; 80(9): 854-63, 2009 Sep.
Article in German | MEDLINE | ID: mdl-19458915

ABSTRACT

INTRODUCTION: Tumors of the carotid body are rare paragangliomas (incidence 0.012%) originating from sympathetic fibres of the carotid bifurcation. Growth is slow and they frequently become symptomatic through local mechanical compression of neighboring vascular and neural structures. The aim of this study is to present the diagnosis, therapy and course in patients with a carotid body tumor treated at our department of the Düsseldorf University Hospital and to discuss rates of recurrence and also dignity during the long-term follow-up. PATIENTS AND METHODS: Included in this retrospective study were all patients treated for a carotid body tumor between January 1988 and June 2008. At follow-up examination the current history was recorded and a physical examination, sonography and duplex sonography were carried out. Furthermore each patient completed the questionnaires QLQ-C30 of the European Organisation for Research and Treatment of Cancer (EORTC) and the module for head and neck QLQ-H&N35 to assess quality of life. RESULTS: In our collective of 36 patients consisting of 13 men (36%) and 23 women (64%) with an average age of 48.33 years (range 17-78 years), 16 patients presented with a local neck swelling and 5 patients each had difficulties swallowing or hoarseness, respectively. Preoperatively Horner's syndrome was found in one patient. A total of 22 tumors were found on the right side of the neck (52.38%), 20 were found on the left side (47.62%) and 6 patients showed a bilateral carotid body tumor (16.67%), 3 of which were bilaterally excised. The other 3 patients are still under surveillance without surgery. Altogether surgery of 39 carotid body tumors was performed in 36 patients. In all 39 cases (primary surgery n=34, recurrence surgery n=5) the tumors were macroscopically excised in toto. Parts of the vagus nerve had to be resected in 3 patients (7.69% Shamblin type II n=1, Shamblin type III n=1) and resection of blood vessels was necessary during 10 operations. The survival rate after 1 year was 100%, after 2 years 96.3% and after 5 years 92.6%. A local recurrence was diagnosed in 2 patients (5.13%). In one patient a second operation was necessary and in the other patient there was a non-progressive swelling in the carotid bifurcation which had existed for 14 years and which was conservatively left untreated. Peripheral neural lesions could be found in 12% (3/25) at long-term follow-up. None of the patients showed evidence of local or remote metastasization of a carotid body tumor. CONCLUSIONS: Surgical extirpation of carotid body tumors can be regarded as the only curative option with an overall mortality of 0%. Morbidity is low when applying vascular surgical techniques (2.56% for central lesions). The incidence of peripheral nervous lesions is high reflecting the radicality of the resection (64.10%) but is outweighed by the benefits. In the long-term follow-up the rate of permanent peripheral neural lesions decreased to 12%. Due to a potentially infiltrating and disseminating growth, carotid body tumors should be regarded as semi-malignant and should therefore be indicated for surgery at the time of diagnosis. Whether the incidence of carotid body tumors will rise due to increased routine diagnostic examination of the head and neck region using sonography and tomography remains to be seen.


Subject(s)
Carotid Body Tumor/surgery , Adolescent , Adult , Aged , Carotid Body/pathology , Carotid Body Tumor/mortality , Carotid Body Tumor/pathology , Cranial Nerve Injuries/etiology , Female , Follow-Up Studies , Humans , Lymph Nodes/pathology , Lymphatic Metastasis/pathology , Male , Middle Aged , Neoplasm Invasiveness/pathology , Neoplasm Recurrence, Local/mortality , Neoplasm Recurrence, Local/pathology , Neoplasm Recurrence, Local/surgery , Postoperative Complications/etiology , Postoperative Complications/mortality , Postoperative Complications/surgery , Reoperation , Retrospective Studies , Survival Rate , Ultrasonography , Young Adult
10.
Zentralbl Chir ; 134(4): 338-44, 2009 Aug.
Article in German | MEDLINE | ID: mdl-19337964

ABSTRACT

BACKGROUND: In 2001 Leschi et al. published a new method to improve perfusion of the superior mesenteric artery (SMA) in operative therapy of acute and chronic visceral ischemia. They presented a retrograde aorto-mesenteric bypass following an arcuate course behind the left renal pedicle. Due to the intricate correct anatomic description of this vascular reconstruction this loop bypass was named the "French bypass". PATIENTS AND METHODS: In our department 84 patients underwent surgery because of an acute or chronic visceral ischemia between January 2002 and December 2007. Out of these patients 27 received a "French bypass". The pre-, intra-, and postoperative data were collected from the patient hospital files retrospectively. The follow-up consisted of a review of the patient history and clinical findings in an outpatient setting, combined with a duplex sonography of the visceral arteries. RESULTS: The group of 27 patients had an average age of 55.0 years: (range: 29-81 years) and consisted of 21 women (78.6 %) and 6 men (21.4 %). The cardinal symptom of all patients was abdominal pain of variable intensities. 14 patients complained about an increased pain post ingestion (abdominal angina) and 12 patients about an involuntary loss of weight. Bypass material was autologous saphenous vein in 18 patients (66.7 %) and in 9 patients (33.3 %) an 8-mm ring-enforced PTFE prosthesis. Apart from 10 patients who only received the French bypass, we performed comprehensive visceral revascularisations in 12 patients. Overall hospital mortality was 18.5 %; 4 out of the 5 deceased patients had undergone surgery due to acute visceral ischemia. The mortality of patients with acute visceral ischemia was 30.8 % and of patients with chronic visceral ischemia 7.1 %. Eight patients had a revision before -discharge from hospital (surgery n = 6, interventional n = 2). Primary and secondary patencies of the bypasses of the surviving patients were 54.6 % (12 out of 22 patients) and 81.8 % (18 out of 22 patients), respectively. Concerning the end-point "freedom from abdominal complaints" 14 out of 27 patients (51.9 %) benefited after a mean follow-up of 38.9 months (range: 3-84 months), 7 patients each in the acute and chronic visceral ischemia group. CONCLUSIONS: The implantation of a "French bypass" represents a good option to reconstruct the SMA, combining the advantages of ante- and retrograde visceral bypasses. Furthermore this -bypass procedure allows to reconstruct distal segments of the -superior mesenteric artery in cases when long distance and peripheral stenosis impeded local thromendarterectomy. Perioperative morbidity and mortality are acceptable when the acute clinical situation is taken into account. The long-term benefit for the patients with regard to the prevention of intestinal ischemia and also the freedom from complaints is high.


Subject(s)
Aorta/surgery , Intestines/blood supply , Ischemia/surgery , Mesenteric Artery, Superior/surgery , Mesenteric Vascular Occlusion/surgery , Vascular Surgical Procedures/methods , Adult , Aged , Aged, 80 and over , Angiography , Blood Vessel Prosthesis , Female , Follow-Up Studies , Hospital Mortality , Humans , Image Processing, Computer-Assisted , Imaging, Three-Dimensional , Ischemia/diagnosis , Ischemia/mortality , Kidney/surgery , Magnetic Resonance Angiography , Male , Mesenteric Vascular Occlusion/diagnosis , Mesenteric Vascular Occlusion/mortality , Middle Aged , Polytetrafluoroethylene , Postoperative Complications/diagnosis , Postoperative Complications/surgery , Reoperation , Retrospective Studies , Tomography, X-Ray Computed , Ultrasonography, Doppler, Duplex , Veins/transplantation
11.
FASEB J ; 23(8): 2425-37, 2009 Aug.
Article in English | MEDLINE | ID: mdl-19364762

ABSTRACT

The identity of store-operated calcium (Ca(2+)) entry (SOCE) channels in vascular smooth muscle cells (VSMCs) remains a highly contentious issue. Whereas previous studies have suggested that SOCE in VSMCs is mediated by the nonselective transient receptor potential canonical (TRPC) 1 protein, the identification of STIM1 and Orai1 as essential components of I(CRAC), a highly Ca(2+)-selective SOCE current in leukocytes, has challenged that view. Here we show that cultured proliferative migratory VSMCs isolated from rat aorta (called "synthetic") display SOCE with classic features, namely inhibition by 2-aminoethoxydiphenyl borate, ML-9, and low concentrations of lanthanides. On store depletion, synthetic VSMCs and A7r5 cells display currents with characteristics of I(CRAC). Protein knockdown of either STIM1 or Orai1 in synthetic VSMCs greatly reduced SOCE, whereas Orai2, Orai3, TRPC1, TRPC4, and TRPC6 knockdown had no effect. Orai1 knockdown reduced I(CRAC) in synthetic VSMCs and A7r5 cells. Synthetic VSMCs showed up-regulated STIM1/Orai1 proteins and SOCE compared with quiescent freshly isolated VSMC. Knockdown of STIM1 and Orai1 inhibited synthetic VSMC proliferation and migration, whereas STIM2, Orai2, and Orai3 knockdown had no effect. To our knowledge, these results are the first to show I(CRAC) in VSMCs and resolve a long-standing controversy by identifying CRAC as the elusive VSMC SOCE channel important for proliferation and migration.


Subject(s)
Calcium Channels/metabolism , Calcium Signaling , Membrane Glycoproteins/metabolism , Myocytes, Smooth Muscle/cytology , Myocytes, Smooth Muscle/physiology , Animals , Calcium Channels/drug effects , Calcium Channels/genetics , Cell Movement/physiology , Cell Proliferation , Cells, Cultured , Membrane Glycoproteins/antagonists & inhibitors , Membrane Glycoproteins/genetics , Muscle, Smooth, Vascular/cytology , Muscle, Smooth, Vascular/drug effects , Muscle, Smooth, Vascular/physiology , Myocytes, Smooth Muscle/drug effects , ORAI1 Protein , Patch-Clamp Techniques , RNA, Small Interfering/genetics , Rats , Stromal Interaction Molecule 1 , Thapsigargin/pharmacology
12.
Circ Res ; 103(11): 1289-99, 2008 Nov 21.
Article in English | MEDLINE | ID: mdl-18845811

ABSTRACT

Recent breakthroughs in the store-operated calcium (Ca(2+)) entry (SOCE) pathway have identified Stim1 as the endoplasmic reticulum Ca(2+) sensor and Orai1 as the pore forming subunit of the highly Ca(2+)-selective CRAC channel expressed in hematopoietic cells. Previous studies, however, have suggested that endothelial cell (EC) SOCE is mediated by the nonselective canonical transient receptor potential channel (TRPC) family, TRPC1 or TRPC4. Here, we show that passive store depletion by thapsigargin or receptor activation by either thrombin or the vascular endothelial growth factor activates the same pathway in primary ECs with classical SOCE pharmacological features. ECs possess the archetypical Ca(2+) release-activated Ca(2+) current (I(CRAC)), albeit of a very small amplitude. Using a maneuver that amplifies currents in divalent-free bath solutions, we show that EC CRAC has similar characteristics to that recorded from rat basophilic leukemia cells, namely a similar time course of activation, sensitivity to 2-aminoethoxydiphenyl borate, and low concentrations of lanthanides, and large Na(+) currents displaying the typical depotentiation. RNA silencing of either Stim1 or Orai1 essentially abolished SOCE and I(CRAC) in ECs, which were rescued by ectopic expression of either Stim1 or Orai1, respectively. Surprisingly, knockdown of either TRPC1 or TRPC4 proteins had no effect on SOCE and I(CRAC). Ectopic expression of Stim1 in ECs increased their I(CRAC) to a size comparable to that in rat basophilic leukemia cells. Knockdown of Stim1, Stim2, or Orai1 inhibited EC proliferation and caused cell cycle arrest at S and G2/M phase, although Orai1 knockdown was more efficient than that of Stim proteins. These results are first to our knowledge to establish the requirement of Stim1/Orai1 in the endothelial SOCE pathway.


Subject(s)
Calcium Channels/physiology , Calcium/physiology , Endothelium, Vascular/physiology , Membrane Glycoproteins/physiology , TRPC Cation Channels/physiology , Animals , Calcium Channels/genetics , Cell Division , DNA Primers , Endothelium, Vascular/cytology , Endothelium, Vascular/drug effects , Humans , Leukemia, Basophilic, Acute/physiopathology , Membrane Glycoproteins/genetics , Membrane Proteins/genetics , Membrane Proteins/physiology , Neoplasm Proteins/genetics , Neoplasm Proteins/physiology , ORAI1 Protein , Patch-Clamp Techniques , RNA, Messenger/genetics , RNA, Small Interfering/genetics , Rats , Stromal Interaction Molecule 1 , Thapsigargin/pharmacology , Umbilical Veins
13.
J Neurochem ; 106(6): 2449-62, 2008 Sep.
Article in English | MEDLINE | ID: mdl-18624925

ABSTRACT

Microglia are the resident immune cells of the CNS, which are important for preserving neural tissue functions, but may also contribute to neurodegeneration. Activation of these cells in infection, inflammation, or trauma leads to the release of various toxic molecules, including reactive oxygen species (ROS) and the excitatory amino acid glutamate. In this study, we used an electrophysiologic approach and a D-[(3)H]aspartate (glutamate) release assay to explore the ROS-dependent regulation of glutamate-permeable volume-regulated anion channels (VRACs). Exposure of rat microglia to hypo-osmotic media stimulated Cl(-) currents and D-[(3)H]aspartate release, both of which were inhibited by the selective VRAC blocker, DCPIB. Exogenously applied H(2)O(2) potently increased swelling-activated glutamate release. Stimulation of microglia with zymosan triggered production of endogenous ROS and strongly enhanced glutamate release via VRAC in swollen cells. The effects of zymosan were attenuated by the ROS scavenger, MnTMPyP, and by two inhibitors of NADPH oxidase (NOX), diphenyliodonium and thioridazine. However, zymosan-stimulated glutamate release was insensitive to other NOX blockers, apocynin and HEBSF. This pharmacologic profile pointed to the potential involvement of apocynin-insensitive NOX4. Using RT-PCR we confirmed that NOX4 is expressed in rat microglial cells along with NOX1 and NOX2. To check for potential involvement of phagocytic NOX2, we stimulated this isoform using protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate or inhibited it with the broad spectrum PKC blocker, Gö6983. Both agents potently modulated endogenous ROS production by NOX2 but not VRAC activity. Taken together, these data suggest that the anion channel VRAC may contribute to microglial glutamate release and that its activity is regulated by endogenous ROS originating from NOX4.


Subject(s)
Excitatory Amino Acids/metabolism , Microglia/metabolism , NADPH Oxidases/metabolism , Voltage-Dependent Anion Channels/metabolism , Zymosan/pharmacology , Animals , Animals, Newborn , Aspartic Acid/metabolism , Cells, Cultured , Encephalitis/metabolism , Enzyme Inhibitors/pharmacology , Glutamic Acid/metabolism , Isoenzymes/drug effects , Isoenzymes/metabolism , Microglia/drug effects , NADPH Oxidase 4 , NADPH Oxidases/drug effects , Oxidants/pharmacology , Oxidative Stress/drug effects , Oxidative Stress/physiology , Protein Kinase C/drug effects , Protein Kinase C/metabolism , Rats , Rats, Sprague-Dawley , Reactive Oxygen Species/metabolism , Voltage-Dependent Anion Channels/drug effects
14.
J Physiol ; 572(Pt 3): 677-89, 2006 May 01.
Article in English | MEDLINE | ID: mdl-16527858

ABSTRACT

Ubiquitously expressed volume-regulated anion channels (VRACs) are chloride channels which are permeable to a variety of small organic anions, including the excitatory amino acids (EAAs) glutamate and aspartate. Broad spectrum anion channel blockers strongly reduce EAA release in cerebral ischaemia and other pathological states associated with prominent astrocytic swelling. However, it is uncertain whether VRAC serves as a major pathway for EAA release from swollen cells. In the present study, we measured swelling-activated release of EAAs as D-[3H]aspartate efflux, and VRAC-mediated Cl- currents by whole-cell patch clamp in cultured rat astrocytes. We compared the pharmacological profiles of the swelling-activated EAA release pathway and Cl- currents. The expression of candidate Cl- channels was confirmed by RT-PCR. The maxi Cl- channel (p-VDAC) blocker Gd3+, the ClC-2 inhibitor Cd2+, and the MDR-1 blocker verapamil did not affect EAA release or VRAC currents. An antagonist of calcium-sensitive Cl- channels (CaCC), niflumic acid, had little effect on EAA release and only partially inhibited swelling-activated Cl- currents. The phorbol ester PDBu, which blocks ClC-3-mediated Cl- currents, had no effect on VRAC currents and up-regulated EAA release. In contrast, DCPIB, which selectively inhibits VRACs, potently suppressed both EAA release and VRAC currents. Two other relatively selective VRAC inhibitors, tamoxifen and phloretin, also blocked the VRAC currents and strongly reduced EAA release. Taken together, our data suggest that (i) astrocytic volume-dependent EAA release is largely mediated by the VRAC, and (ii) the ClC-2, ClC-3, ClC-4, ClC-5, VDAC, CaCC, MDR-1 and CFTR gene products do not contribute to EAA permeability.


Subject(s)
Astrocytes/metabolism , Chloride Channels/physiology , Chlorine/metabolism , Excitatory Amino Acid Agents/administration & dosage , Excitatory Amino Acids/metabolism , Ion Channel Gating/physiology , Water-Electrolyte Balance/physiology , Animals , Animals, Newborn , Astrocytes/cytology , Cell Size , Cells, Cultured , Ion Channel Gating/drug effects , Osmotic Pressure , Rats , Rats, Sprague-Dawley , Water-Electrolyte Balance/drug effects
15.
J Physiol ; 549(Pt 3): 749-58, 2003 Jun 15.
Article in English | MEDLINE | ID: mdl-12702740

ABSTRACT

Whole-cell recordings showed that, in mouse mammary C127 cells transfected with the full genome of the bovine papilloma virus (BPV), a hypotonic challenge induced the activation of outwardly rectifying Cl- currents with a peak amplitude 2.7 times greater than that in control C127 cells. Cell-attached single-channel recordings showed that BPV-induced augmentation of the peak amplitude of the whole-cell current could not chiefly be explained by a small increase (1.2 times) in unitary conductance. There was no difference between control and BPV-transfected cells in the osmotic cell swelling rate, and hence, osmotic water permeability. However, a plot of the whole-cell current density as a function of cell volume, which was measured simultaneously, showed that the BPV-transfected cells had a strikingly greater volume sensitivity than control cells. Since the E5 protein of BPV has been reported to induce constitutive activation of the epidermal growth factor (EGF) receptor and platelet-derived growth factor (PDGF) receptor in a variety of cell lines including C127 cells, effects of the growth factors on volume-sensitive outwardly rectifying (VSOR) Cl- currents were examined in C127 cells. Application of PDGF peptides failed to affect the Cl- currents in control and BPV-transfected cells, although C127 cells are known to endogenously express PDGF receptors. In contrast, EGF peptides significantly increased the VSOR Cl- current in control cells. However, they failed to induce further augmentation of the current in BPV-transfected cells. VSOR Cl- currents were inhibited by tyrphostin B46, an inhibitor of the EGF receptor tyrosine kinase, in both control and BPV-transfected cells. The IC50 value in BPV-transfected cells (12 micro M) was lower than that in control cells (31 micro M). However, the VSOR Cl- currents in both cell types were insensitive to tyrphostin AG1296, an inhibitor of the PDGF receptor tyrosine kinase. The rate of regulatory volume decrease (RVD) was markedly diminished by tyrphostin B46 but not significantly affected by tyrphostin AG1296. We thus conclude that the EGF receptor tyrosine kinase upregulates the activity of the VSOR Cl- channel, mainly by enhancing the volume sensitivity.


Subject(s)
Chloride Channels/physiology , ErbB Receptors/biosynthesis , Mammary Glands, Animal/cytology , Up-Regulation/physiology , Algorithms , Animals , Cell Line , Cell Membrane Permeability/physiology , Cell Size/physiology , Chloride Channels/drug effects , Chloride Channels/genetics , Cystic Fibrosis Transmembrane Conductance Regulator/biosynthesis , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Electrophysiology , ErbB Receptors/genetics , Female , Mammary Glands, Animal/drug effects , Membrane Potentials/physiology , Mice , Osmotic Pressure , Patch-Clamp Techniques , Transfection , Up-Regulation/drug effects
16.
Pflugers Arch ; 445(2): 177-86, 2002 Nov.
Article in English | MEDLINE | ID: mdl-12457238

ABSTRACT

Transient expression of wild-type human cystic fibrosis transmembrane conductance regulator (CFTR) in HEK293T cells resulted in a profound decrease in the amplitude of volume-sensitive outwardly rectifying Cl- channel (VSOR) current without changing the single-channel amplitude. This effect was not mimicked by expression of the DeltaF508 mutant of CFTR, which did not reach the plasma membrane. The VSOR regulation by CFTR was not affected by G551D mutation at first nucleotide-binding domain (NBD1), which is known to impair CFTR interaction with the outwardly rectifying chloride channel, ORCC, epithelial amiloride-sensitive Na-channel, ENaC, and renal potassium channel, ROMK2. The CFTR-VSOR interaction was insensitive to the deletion mutation, DeltaTRL, which is known to impair CFTR-PDZ domain binding. In contrast, the G1349D mutant, which impairs ATP binding at NBD2, effectively abolished the down-regulatory effect of CFTR. Furthermore, the K1250M mutation at the Walker A motif and the D1370N mutation at the Walker B motif, both known to impair ATP hydrolysis at NBD2, completely abolished the VSOR regulation by CFTR. Thus, we conclude that an ATP-hydrolysable conformation of NBD2 is essential for the regulation of the VSOR by the CFTR protein, and that VSOR is a first channel regulated by CFTR through its NBD2.


Subject(s)
Chloride Channels/metabolism , Cystic Fibrosis Transmembrane Conductance Regulator/physiology , Adenosine Triphosphate/metabolism , Cell Line , Chloride Channels/physiology , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Down-Regulation , Gene Deletion , Humans , Hydrolysis , Molecular Conformation , Nucleotides/metabolism , Patch-Clamp Techniques , Protein Structure, Tertiary/physiology
17.
Majalah Obstet Ginekol Indones ; 8(1): 44-51, 1982 Jan.
Article in Indonesian | MEDLINE | ID: mdl-12339013

ABSTRACT

PIP: The perinatal mortality rate remains high in developing countries at the current time. Perinatal mortality was reviewed in the Gatot Soebroto Army Central Hospital during the period January 1, 1979-December 31, 1980. During this period, there were 2813 deliveries with 37 stillbirths (1.3%), 81 early neonatal deaths (2.9%), and 118 perinatal deaths (4.2%). In this paper, the relationship between perinatal mortality and the birthweight, type of delivery, antenatal care, and mother's age and parity were reviewed. The Gatot Soebroto Army Central Hospital, in addition to providing services to members of the Armed Forces of the Republic of Indonesia, also accepts patients from outside the Armed Forces. (author's modified)^ieng


Subject(s)
Fetal Death , Infant Mortality , Mortality , Socioeconomic Factors , Asia , Asia, Southeastern , Birth Weight , Delivery of Health Care , Delivery, Obstetric , Demography , Developing Countries , Economics , Indonesia , Maternal Age , Parity , Population , Population Dynamics
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